An early disclosure of the conversion of hydrocarbons of the paraffinic type by bacterial action is described in U.S. Pat. No. 2,396,900. The method described in that patent converts normally gaseous paraffinic hydrocarbons into heavy, waxy, oxygenated organic compounds by contacting the hydrocarbons in the presence of oxygen with an aqueous nutrient solution inoculated with hydrocarbon consuming bacteria of the group consisting of Bacillus methanicus and Bacillus ethanicus. The patent describes a continuous process carried out in apparatus similar to a bubble cap tower. The patent speaks of the bacteria consuming the hydrocarbons. It describes what goes on in the patented process as the synthesis, from light hydrocarbons, of oxygenated organic compounds of various molecular weights, from low boiling alcohols to waxy acids, esters and alcohols. When the reaction is permitted to proceed to completion, the product is a predominantly heavy waxy body composed of fatty acids and esters thereof, that may be readily saponified.
A later U.S. Pat. No. 3,622,465, describes a process in which the microorganism Arthrobacter simplex utilizes C.sub.3 -C.sub.18 straight chain hydrocarbons as a principal source of assimilable carbon and energy to produce single cell protein. The fermentation is carried out, in one embodiment of the invention, on a continuous basis in a sieve plate column, using liquified propane gas as the hydrocarbon.
In my-copending patent application, Ser. No. 160,273, filed June 17, 1980, a continuous process is described comprising establishing a series of separate but interconnected sequential contact zones, flowing a liquid composition comprising a biocatalyst through each of said zones successively from a liquid inlet zone to a liquid outlet zone, flowing an oxidizing gas through each of said zones successively from a gas inlet zone to a gas outlet zone to a gas outlet zone, in intimate, countercurrent contact with the flowing liquid in each of said zones, flowing an organic substrate successively through each of said zones in intimate, reactive contact with said gas and with the liquid composition containing said biocatalyst, and recovering liquid effluent discharged from the liquid outlet zone and gas effluent discharged from the gas outlet zone, the recovered effluents comprising at least some of the oxidizable organic substrate converted to a more advanced state of oxidization. In a preferred embodiment, the gas-liquid contact apparatus in which the process is carried out in a bubble cap tower. In a very preferred mode of practice of the process, C.sub.2 -C.sub.4 n-alkenes and butadiene, particularly propylene, are converted to the corresponding epoxides.
The biocatalytic oxidation reactions, with which the present invention is concerned, have been described in recent literature. In such reactions a biocatalyst is utilized in the presence of oxygen for the conversion of gaseous hydrocarbons into their respective corresponding alcohols, aldehydes, ketones and/or epoxides. Several suggestions have been made in the literature that such processes could be practiced on a continuous basis, but no details were reported for a practical continuous process except for my co-pending patent application Ser. No. 160,273, filed June 17, 1980.
The discovery and isolation of certain methylotrophic microorganisms strains, that grow well under aerobic conditions in a culture medium in the presence of methane as the major carbon and energy source, are reported in U.K. patent application GB 2,018,822 A, published Oct. 24, 1979, which is incorporated herein by reference. These methane-grown microbial cells possess a high content protein. The cells, or enzyme preparations derived from the cells, are said to be useful in converting oxidizable substrates to oxidation products. In particular, C.sub.1 -C.sub.6 alkanes can be converted to alcohols, such as methane to methanol; C.sub.3 -C.sub.6 alkanes can be converted to the corresponding secondary alcohols and methyl ketones; C.sub.3 -C.sub.6 secondary alcohols can be converted to the corresponding methyl ketones; and cyclic hydrocarbons can be converted to cyclic hydrocarbyl alochols, such as cyclohexane to cyclohexanol; and C.sub.2 -C.sub.4 alkenes selected from the group consisting of ethylene, propylene, butene-1 and butadiene, can be converted to 1,2-epoxides.
Cell-free extracts of certain of these hydrocarbon-utilizing microbes, including bacteria and yeasts, contain a nicotinamide adenine dinucleotide (NAD) dependent secondary alcohol dehydrogenase (SADH). This enzyme specifically and stoichiometrically oxidizes C.sub.3 -C.sub.6 secondary alcohols, such as 2-propanol and 2-butanol, to their corresponding ketones.
A process for the epoxidation of C.sub.2 -C.sub.4 alpha olefins and dienes, through the action of a particular kind of biocatalyst in the presence of oxygen, is described in U.K. patent application GB 2,019,390 A, which is incorporated herein by reference. The biocatalyst is a particulate fraction of the microorganism, or an enzyme preparation derived therefrom. The microorganisms are cultivated in a nutrient medium furnishing oxygen and methane or dimethyl ether. The preferred microorganisms are obligative or facultative methylotrophs. Several particularly preferred strains are identified.
In U.K. patent application GB 2,018,772 A, published Oct. 24, 1979, a process is disclosed for the production of ketones or secondary alcohols from C.sub.3 -C.sub.6 alkanes, and ketones from C.sub.3 -C.sub.6 secondary alcohols. The process is conducted under aerobic conditions with resting microbial cells derived from a methyloptophic microoroganism, or with an enzyme preparation derived from such cells. The microorganism is one that has been grown under aerobic conditions in a nutrient medium containing a C.sub.1 -compound and energy source which is an inducer for the enzyme(s) responsible for producing the ketones. The C.sub.1 compound may be, for example, methane, methanol, dimethyl ether, methylamine, methyl formate, or methyl carbonate. The term microorganism includes bacteria, protozoa, yeasts, filamentous fungi, and actinomycetes. Yeast cells, grown as referred to, are shown as useful in aerobically converting C.sub.3 -C.sub.6 secondary alochols. The preparation, isolation and purification of a C.sub.3 -C.sub.6 secondary alcohol dehydrogenase is also described.
The oxidation of alkanes having from 5 to 16 carbon atoms, or of aliphatic alcohols having from 3 to 8 carbon atoms, or cyclic organic compounds, utilizing a biocatalyst, is described in U.K. patent application GB 2,024,205 A, published Jan. 9, 1980, which is also incorporated herein by reference. In the process described in this application, the biocatalyst may be a culture of a methane-utilizing bacterium of the species Methylosinus trichosporium or an extract thereof containing a methane oxidizing system.
U.K. patent application 27,886, filed July 4, 1977, and supplemented May 25, 1978, describes the liquid phase oxidation of straight chain alkanes having more than 3 and less than 9 carbon atoms, of alkenes, and of cyclic organic compounds, utilizing as the biocatalyst a culture of a methane oxidizing bacterium or an extract thereof containing a methane oxidizing system. One of the asserted advantages of this process, when enzyme extracts rather than whole cells were used, is said to be the regeneration in situ of cofactors or other biochemical species required for the enzymatic reaction. While the examples describe, and the specification emphasizes, liquid phase oxidation in which a homogenous catalyst is used, one way of carrying out the process that is suggested as a possibility, involves immobilizing such cells on a suitable support material such as glass beads or gel matrix, to form an immobilized enzyme preparation based on the use of cells as the enzyme source. This immobilized enzyme preparation, it is said, may be maintained in a packed or fluidized bed in a suitable contactor. The disclosure of this patent application is also incorporated herein by reference.
These published British patent applications include many references to the pertinent specific literature. A few such items are described below and are incorporated herein by reference.
Hutchinson, Whittenbury and Dalton (J. Theor. Biol., 58 325-335 (1976) "A Possible Role of Free Radicals in the Oxidation of Methane by Methylococcus Capsulatus" and Colby and Dalton (J. Biochem., 157, 495-497 (1976) "Some Properties of a Soluble Methane Mono-Oxygenase from Methylococcus Capsulatus Strain Bath" reported that ethylene is oxidized by the soluble methane mono-oxygenase from Methylococcus Capsulatus Strain Bath. The latter investigators reported that the "particulate membrane preparations" of Methylococcus capsulatus Strain Bath did not have methane-oxygenase activity as determined by the bromomethane disappearance test.
Cerniglia, Blevens and Perry, (Applied and Environmental Microbiology, 32 (6) 764-768 (1976) "Microbial Oxidation and Assimilation of Propylene" described the oxidation of propylene by microorganisms to the corresponding alcohols and carboxylic acids.
Most recently, Colby, Stirling and Dalton (J. Biochem., 165, 395-402 (Aug. 1977) "The Soluble Methane Mono-Oxygenase of Methylococcus capsulates (Bath) its Ability to Oxygenate n-Alkenes, Ethers, and Alicyclic Aromatic and Heterocyclic Compounds") disclosed that the soluble fraction of Methylococcus Capsulatus Strain Bath is a very non-specific oxygenase in that it oxidizes alkanes to alcohols, alkenes to 1,2-epoxides, dimethylether to ethanol and ethanal, styrene to styrene epoxide and pyridine to pyridine N-oxide.
On the basis of .sup.18 0.sub.2 incorporation from .sup.18 0.sub.2 into the cellular constituents of Pseudomonas methanica, Leadbetter and Foster (Nature, 184: 1428-1429 (1959) "Incorporation of Molecular Oxygen in Bacterial Cells Utilizing Hydrocarbons for Growth" suggested that the initial oxidative attack on methane involves an oxygenase. Higgins and Quayle (J. Biochem., 118, 201-208 (1970) "Oxygenation of Methane by Methane-Grown Pseudomonas methanica and Methanomonas methanooxidans") isolated CH.sub.3.sup.18 OH as the product of methane oxidation when suspensions of Pseudomanas methanica or Methanomonas methanooxidans were allowed to oxidize methane in .sup.18 0.sub.2 enriched atmospheres. The subsequent observation of methane-stimulated NADH oxidation catalyzed by extracts of Methylococcus Capsulatus by Ribbons (J. Bacteriol, 122: 1351-1363 (1975) "Oxidation of C.sub.1 Compounds by Particulate Fractions from Methylococcus Capsulatus: Distribution and Properties of Methane-Dependent Reduced Nicotinamide Adenine Dinucleotide Oxidane") (methane hydroxylase) and Ribbons and Michalover, (FEBS Lett. 11: 41-44 (1970) "Methane Oxidation by Cell-Free Extracts of Methylococcus Capsulatus") or of Methylomonas Methanica by Ferenci (FEBS Lett. 41: 94-98 (1974) "Carbon Monoxide-stimulated Respiration in Methane-Utilizing Bacteria") suggested that the enzyme responsible for this oxygenation is a mono-oxygenase.
Recently, methane monooxygenase systems were partially purified from Methylosinus trichosporium OB3b (Tonge, Harrison and Higgins, J. Biochem., 161: 333-344 (1977) "Purification and Properties of the Methane Monooxygenase Enzyme System from Methylosinus trichosporium OB3b"; and Tonge, Harrison, Knowles and Higgins, FEBS Lett., 58; 293-299 (1975) "Properties and Partial Purification of the Methane-Oxidizing Enzyme System from Methylosinus trichosporium") and Methylococcus Capsulatus (Bath) (Colby and Dalton, J. Biochem., 171: 461-468 (1978) "Resolution of the Methane Mono-Oxygenase of Methylococcus Capsulatus (Bath) into Three Components" and Colby, Stirling and Dalton, J. Biochem., 165: 395-402 (1977) "The Soluble Methane Mono-Oxygenase of Methylococcus Capsulatus (Bath) Its Ability to Oxygenate n-Alkanes, n-Alkenes, Ethers, and Alicyclic, Aromatic and Heterocyclic Compounds").
In addition, there are several rather recent literature items of interest, as described below, and each of these is also incorporated herein by reference. These items are described below in chronological order.
Colby and Dalton (Biochem. J., 171, 461-468 (1978)), "Resolution of the Methane Mono-Oxygenase of Methylococcus Capsulatus (Bath) into Three Components", describe the fractionation of the enzyme extract into three fractions by ion exchange chromatography. The authors point out that the soluble enzyme extract itself is capable of oxidizing a variety of alkanes, alkenes, ethers and cyclic compounds. Further work was reported by Stirling, Colby, and Dalton (Biochem. J., 177, 361-364 (1979)), "A Comparison of the Substrate and Electron-Donor Specificities of the Methane Mono-Oxygenase from Three Strains of Methane-Oxidizing Bacteria". The authors concluded that similar methane mono-oxygenases were contained in the three bacteria, Methylosinus Trichosporium, Methylococcus capsulatus (Bath), and Methylomonas methanica, based upon studies made with extracts.
Stirling and Dalton (FEMS Microbiology Letters 5, 315-318 (1979)), "The Fortuitous Oxidation and Cametabolism of Various Carbon Compounds by Whole-Cell Suspensions of Methylococcus capsulatus (Bath)", report that cell suspensions of this organism do not behave in the same manner as extracts, as to oxidizing activity.
More recently, Higgins, Best and Hammond, in a review article (Nature 286, 561-4 (1980)), "New Findings in Methane-Utilizing Bacteria Highlight Their Importance in the Biosphere and Their Commercial Potential", presented a survey of recent developments. They point out that as recently as 1965, methanotrophs were regarded, even by most microbiologists, as obscure, uncooperative, perhaps unimportant microorganisms, as evidenced by the fact that, before 1970, only three species had been isolated and well authenticated. Today it is recognized, they say, that these microorganisms include at best two different types of species. Carbon is incorporated into cell material at the oxidation level of formaldehyde by type I species which use the ribulose monophosphate pathway (Quayle cycle) and in type II species, using the serine pathway, as formaldehyde and carbon dioxide. Such bacteria, either as washed suspensions or in culture, will partially oxidize simple substrate analogues, such as ethene, propane and butane, to the corresponding alcohols, aldehydes and fatty acids. It has been shown that carbon monoxide, ammonia and ethene are also oxidized. The authors also state that a surprisingly vast range of multi-carbon compounds, often not closely related to the natural substrates, are oxidized by methanotrophs. Although the capacity to oxidize is said to differ from species to species, the authors state that "the following types of compounds are oxidized by washed cell suspensions: long-chain alkanes (up to at least hexadecane), alkenes, aromatic and alicyclic hydrocarbons, phenols, long-chain and alicyclic alcohols, pyridine, multi-ring compounds and chlorinated aromatic hydrocarbons. In each case only a limited number of products (sometimes only one) are formed as a result of this unexpected activity, showing that there is, nevertheless, some mechanistic specificity. In some cases the products are simply hydroxylated derivatives, suggesting that a reaction analogous to the oxidation of methane to methanol has occurred. Commonly, there is further oxidation of these hydroxylated compounds to yield aldehydes and carboxylic acids."